CN110982700B

CN110982700B - Polyketide with anti-helicobacter pylori activity and preparation method and application thereof

Info

Publication number: CN110982700B
Application number: CN201911038386.4A
Authority: CN
Inventors: 刘玲; 郭良栋; 郭龙芳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2021-11-12
Anticipated expiration: 2039-10-29
Also published as: CN110982700A

Abstract

The invention discloses a polyketide with anti-helicobacter pylori activity, a preparation method and an application thereof. The compound has good activity on helicobacter pylori drug-resistant strains (H.pylori BHKS 159). The structural formula of the helicobacter pylori resistant active compound provided by the invention is shown as a formula (I). The invention also provides a strain Shsl21 for producing the compound shown in the formula (I), and the preservation number is CGMCC No. 18550. The invention also provides a method for preparing the compound shown in the formula (I) by using the strain Shsl21 and application of the compound shown in the formula (I) in inhibiting H.pylori BHKS159, wherein the compound is suitable for research of a lead compound for resisting helicobacter pylori or preparation of a medicament for resisting helicobacter pylori.

Description

Polyketide with anti-helicobacter pylori activity and preparation method and application thereof

Technical Field

The present invention belongs to the field of polyketide compound technology with activity of resisting helicobacter pylori.

Background

Helicobacter pylori (Helicobacter pylori) is a microaerophilic spiral gram-negative bacterium which can be planted on gastric mucosa for a long time, the infection rate of the Helicobacter pylori reaches over 50% of the global population, China is also a country with higher infection rate, and the infection rate is 40-90% according to statistics. Helicobacter pylori is an important causative agent in the pathogenesis of chronic gastritis, streptococcal disease, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The world health organization/international agency for research on cancer has listed helicobacter pylori as a human class i carcinogen. Therefore, the research and development of high-efficiency and low-toxicity helicobacter pylori medicines are in the forefront.

Disclosure of Invention

The invention aims to provide a compound with anti-helicobacter pylori activity, which has a structural formula shown as a formula I:

the second purpose of the invention is to provide a bacterial strain, which is named as a bacterial strain Shsl21, belongs to Fusarium equiseti (Fusarium equiseti), and is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Siro-1 Hospital of Indormitopsis, Beijing) in 19 months in 2019, and the registration number of the preservation center is CGMCC No. 18550.

A third object of the present invention is to provide a method for preparing the compound, comprising the steps of:

fermenting fusarium equiseti (F.equiseti, CGMCC No.18550) in a solid culture medium to obtain a fermentation product, and extracting, separating and purifying the fermentation product to obtain the compound.

A step of leaching a fermentation product obtained by fermenting the strain Shsl21 with an organic solvent. The organic solvent may be ethyl acetate.

The leaching method comprises the following specific steps: extracting the fermented product with ethyl acetate at room temperature for 3-5d (specifically 4 d). During the leaching process, the ethyl acetate can be replaced every 24 hours, and finally the supernatant of the extracting solution is combined.

The fermentation can specifically adopt the following culture media: 80g of rice was soaked in 120mL of water overnight to obtain a medium. The fermentation conditions may be specifically 25 ℃ and 30 days (sterile culture).

The method for separation and purification comprises the following steps:

sequentially carrying out reduced pressure silica gel column chromatography, reversed phase silica gel chromatography and gel chromatography on the leached product, eluting by using a solution capable of dissolving the compound, and collecting an eluent containing the compound; separating the eluate by reverse phase High Performance Liquid Chromatography (HPLC) to obtain the compound.

It is still another object of the present invention to provide a medicament for anti-helicobacter pylori activity.

The active ingredient of the medicine for resisting helicobacter pylori provided by the invention is the compound.

The application of the compound in preparing the anti-helicobacter pylori medicine also belongs to the protection scope of the invention.

It is still another object of the present invention to provide a method for producing a drug for anti-helicobacter pylori.

The method for preparing the medicament for resisting the helicobacter pylori, which is provided by the invention, is used for preparing the medicament for resisting the helicobacter pylori by taking the compound as an active ingredient.

The helicobacter pylori refers to levofloxacin, clarithromycin and metronidazole multidrug-resistant bacterium H.pyrori BHKS 159.

The invention provides a strain and a known compound, the compound has better anti-helicobacter pylori (H.pylori BHKS159) activity, and is suitable for anti-helicobacter pylori lead compound research or preparation of anti-helicobacter pylori medicaments.

Drawings

FIG. 1 is a drawing of a compound¹H nuclear magnetic resonance spectrum.

FIG. 2 is a drawing of a compound¹³C nuclear magnetic resonance spectrum.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical stores, unless otherwise specified. The quantitative tests in the following examples were all set up for 3 replicates and the results were averaged.

Example 1

Isolation and characterization of Strain Shsl21

Collection and separation of strain Shsl21

A strain named as Shsl21 was isolated from flowers of the northern regional Tung-Hua tree in Guangxi defense harbor in 2015.

The strain Shsl21 is a mangrove endophytic fungus.

II, identification of strain Shsl21

1. Molecular identification

The partial sequence of the 18S ribosomal RNA of the strain Shsl21 is shown in sequence 1 of the sequence table, for example, the sequence similarity of GENBANK ACCESSION NO. MN443741 is the highest, and the similarity is 99%.

According to the above identification results, the strain Shsl21 belongs to Fusarium equiseti (Fusarium equiseti).

Third, preservation of Strain Shsl21

The strain Shsl21 belongs to Fusarium equiseti (Fusarium equiseti), and has been deposited in China general microbiological culture Collection center (CGMCC for short, with the address of CGMCC No. 3 of Beijing city Kogyo No.1 of North Chen West Lu of the Indormiton) in 19 months in 2019, and the preservation number is CGMCC No. 18550.

Example 2

Preparation of the Compounds

PDA culture medium: mixing potato 200g, glucose 20g, agar 15g and water 1000mL, and sterilizing with high pressure steam at 121 deg.C for 30 min.

Seed culture medium: glucose 4.0g, wort 10.0g, yeast extract 4.0g and water 1000mL were mixed well, pH was adjusted to 6.5, 50mL was added to each 250mL Erlenmeyer flask and sterilized at 121 ℃ for 30 min.

Rice culture medium: 80g of rice and 120mL of water were put into a 500mL Erlenmeyer flask, soaked overnight, and autoclaved at 121 ℃ for 30 min.

1 solid fermentation of Strain Shsl21

1.1 activation of Strain Shsl21

Culturing the strain Shsl21 on a PDA plate for 5 days, and after the colony grows to fill the plate, culturing 5 colonies with the length of 1cm²The pellet of the size was inoculated into a 250mL Erlenmeyer flask containing 50mL of seed medium together with the medium, and cultured on a shaker (25 ℃, 200rpm) for 7 days to serve as a seed culture solution.

1.2 solid fermentation of Strain Shsl21

Inoculating 5mL of seed culture solution into a triangular flask containing rice culture medium, and performing aseptic culture at 25 deg.C for 30 d.

2 extraction of the Compound

2.1 Add 1.0L of ethyl acetate to the flask (containing the solid fermentation) obtained in step 1.2, leach at room temperature for 24h, collect the first supernatant.

2.2 Add 1.0L of ethyl acetate again to the Erlenmeyer flask of step 2.1, leach at room temperature for 24h, collect the second supernatant.

2.3 Add 1.0L of ethyl acetate again to the Erlenmeyer flask of step 2.2, leach at room temperature for 24h, collect the third supernatant.

2.4 Add 1.0L of ethyl acetate again to the Erlenmeyer flask of step 2.3, leach at room temperature for 24h, collect the fourth supernatant.

2.5 the first supernatant of step 2.1, the second supernatant of step 2.2, the third supernatant of step 2.3 and the fourth supernatant of step 2.4 are combined and distilled to dryness under reduced pressure to give 14.0g of crude extract.

3. Separation and purification of compounds

3.1 vacuum silica gel column chromatography to obtain the eluent

And (3) carrying out reduced pressure silica gel column chromatography on the crude extract obtained in the step 2.5.

The types of the columns are as follows: 8 × 30 cm;

the fillers are: thin layer chromatography silica gel H and column chromatography silica gel (refined);

the elution process is as follows:

(1) eluting with 1000mL of eluent A (composed of 6 parts by volume of petroleum ether and 4 parts by volume of ethyl acetate) in sequence;

(2) eluting with 1000mL of eluent B (composed of 5 parts by volume of petroleum ether and 5 parts by volume of ethyl acetate);

(3) eluting with 1000mL of eluent C (composed of 4 parts by volume of petroleum ether and 6 parts by volume of ethyl acetate);

(4) eluting with 1000mL of eluent D (composed of 3 parts by volume of petroleum ether and 7 parts by volume of ethyl acetate);

(5) eluting with 1000mL of eluent E (consisting of 2 parts by volume of petroleum ether and 8 parts by volume of ethyl acetate);

the flow rate is 50mL/min, and the eluent A-E is collected after passing through the column.

3.2 reverse silica gel chromatographic separation of the chromatographic eluent to obtain reverse eluent

The parameters of the reverse silica gel chromatographic separation are as follows:

the types of the columns are as follows: 3 × 60 cm;

the fillers are: c18;

the mobile phase was 500mL eluent (composed of 6 parts by volume methanol and 4 parts by volume water) at a flow rate of 0.5 mL/min;

collecting the eluent in reverse direction after passing through the column.

3.3 subjecting the reverse eluent to gel chromatography to obtain a gel eluent

The parameters of the gel chromatographic separation are as follows:

the types of the columns are as follows: 3 × 60 cm;

the fillers are: sephadex LH-20;

the mobile phase is a methanol organic solvent with the percentage content of 100 percent, and the flow rate is 0.5 mL/min;

collecting the gel eluent with the retention volume of 80-100 mL.

3.4 reverse phase HPLC separation is carried out on the gel eluent to obtain HPLC eluent

The parameters of the reverse phase HPLC separation are as follows:

the types of the columns are as follows: a Kramosil C18 column (10 μm; 10X 250 mm);

the elution process is as follows: eluting with 57-59% methanol water solution for 30min at flow rate of 2 mL/min.

The collection retention time is 20.6-21.0min (retention time of peak value t)_R20.8min) of the eluate after passing through the column.

3.5 distillation under reduced pressure of the HPLC eluent collected in step 3.4 to dryness gives 5.5mg of product.

4 characterization of the Compounds

And (3) carrying out organic mass spectrometry and nuclear magnetic resonance spectrum analysis on the product of the step 3.5.

The characterization data of the product are as follows:

yellow needle-shaped;

the molecular formula is as follows: c₁₄H₂₀O₆(ii) a Molecular weight: 284;

¹the H nuclear magnetic resonance spectrum is shown in figure 1,¹³the C NMR spectrum is shown in FIG. 2;

based on the above characterization data and the physicochemical data and profile of the above compound, the product of step 3.5 is a compound of formula (I).

Example 3

Inhibition of H.pylori BHKS159 by the compound of formula (I)

The minimum inhibitory concentration (MIC, 100 μ L system) of the compound represented by formula (I) against h.pylori BHKS159 was examined by microdilution.

Parallel experiments were performed with metronidazole (aladin) as a positive control for the compounds.

The bacteria used in this example were: helicobacter pylori (h. pylori BHKS 159).

The experiment comprises the following specific steps:

1. preparation of MIC plates

Adding a brain heart extract culture medium (BHI) (specific components comprise peptone, dehydrated calf brain extract powder, dehydrated calf heart extract powder, sodium chloride, glucose and disodium hydrogen phosphate, and the pH value is 7.4) to 173.6 mu L of the BHI in the 1 st hole, adding 6.4 mu L of a compound to be detected, and diluting to the 7 th hole in a multiple ratio; no addition was made in well 8, and 90. mu.L of the medium was retained as a control with no addition of test compound.

2. Preparation of bacterial liquid

Taking helicobacter pylori (H.pyrori BHKS159) growing on a solid plate in a logarithmic phase, preparing a bacterial suspension by using a BHI culture medium, and adjusting the concentration OD₆₀₀Is 0.3 (1X 10)⁸CFU/mL), 10-fold dilution at 1X 10⁷CFU/mL, spare.

3. Inoculated bacterial liquid

Adding 10 μ L into 1-8 th well (the concentration of bacteria liquid in each well is about 1.0 × 10)⁶CFU/mL). And culturing for 72h to judge the result. The compound concentrations in wells 1 to 7 were 64, 32, 16, 8, 4, 2, 1. mu.g/mL, respectively.

4. Result judgment

The MIC was taken as the lowest test compound concentration that completely inhibited bacterial growth in the wells.

The test is meaningful when the bacteria grow significantly in the 8 th well (i.e., no antibiotic) of the positive control well.

When a single jump hole occurs in the microdilution method, the highest concentration of compound that inhibits bacterial growth should be recorded. If a plurality of jump holes appear, the result should not be reported, and the test needs to be repeated.

The bacteriostatic action of the compound shown in the formula (I) on helicobacter pylori (H.pylori BHKS159) needs to be repeated for 3 times.

The MIC value of the compound represented by the formula (I) against helicobacter pylori (H.pylori BHKS159) was 8. mu.g/mL.

The MIC value of metronidazole to helicobacter pylori (h. pylori BHKS159) was greater than 16 μ g/mL.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> polyketide with anti-helicobacter pylori activity, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 481

<212> DNA

<213> Fusarium (Fusarium equiseti)

<400> 1

acggcgtggc cgcgacgatc accagtaacg aggtgtatga ttactacgct atggaagctc 60

gacgtgaccg ccaatcgatt tggggaacgc gggttaccgc gagtcccaac accaagctga 120

gcttgagggt tgaaatgacg ctcgaacagg catgcccgcc agaatactgg cgggcgcaat 180

gtgcgttcaa agattcgatg attcactgaa ttctgcaatt cacattactt atcgcatttt 240

gctgcgttct tcatcgatgc cagaaccaag agatccgttg ttgaaagttt tgatttattt 300

gtttgtttta ctcagaagtt ccactaaaaa cagagtttag gggtcctcgg gcgggccgtc 360

ccgttttacg gggcgcgggc tgatccgccg aggcaacgta taggtatgtt cacaggggtt 420

tgggagttgt aaactcggta atgatccctc cgctggttca ccaacggaga ccttgttacg 480

a 481

Claims

1. A method for preparing a compound with anti-helicobacter pylori activity is characterized by comprising the following steps:

activated strain Shsl 21;

a solid fermentation strain Shsl 21;

leaching the solid fermentation product with ethyl acetate to obtain a crude extract;

performing reduced pressure silica gel column chromatography to obtain eluate;

separating with reverse silica gel chromatography to obtain reverse eluate;

separating with gel chromatography to obtain gel eluate;

separating by reverse phase HPLC to obtain HPLC eluate containing compound with helicobacter pylori resisting activity;

the preservation number of the strain Shsl21 is CGMCC No. 18550;

the structural formula of the compound with the activity of resisting helicobacter pylori is shown as the following formula:

2. the use of a compound prepared by the process of claim 1 for the manufacture of a medicament for inhibiting helicobacter pylori activity.

3. The use according to claim 2, wherein the minimum concentration of the compound to inhibit bacterial growth is 8 μ g/mL.